RESUMO
The availability of high-frequency, real-time measurements of the concentrations of specific metabolites in cell culture systems will enable a deeper understanding of cellular metabolism and facilitate the application of good laboratory practice standards in cell culture protocols. However, currently available approaches to this end either are constrained to single-time-point and single-parameter measurements or are limited in the range of detectable analytes. Electrochemical aptamer-based (EAB) biosensors have demonstrated utility in real-time monitoring of analytes in vivo in blood and tissues. Here, we characterize a pH-sensing capability of EAB sensors that is independent of the specific target analyte of the aptamer sequence. We applied this dual-purpose EAB to the continuous measurement of pH and phenylalanine in several in vitro cell culture settings. The miniature EAB sensor that we developed exhibits rapid response times, good stability, high repeatability, and biologically relevant sensitivity. We also developed and characterized a leak-free reference electrode that mitigates the potential cytotoxic effects of silver ions released from conventional reference electrodes. Using the resulting dual-purpose sensor, we performed hourly measurements of pH and phenylalanine concentrations in the medium superfusing cultured epithelial tumor cell lines (A549, MDA-MB-23) and a human fibroblast cell line (MRC-5) for periods of up to 72 h. Our scalable technology may be multiplexed for high-throughput monitoring of pH and multiple analytes in support of the broad metabolic qualification of microphysiological systems.
RESUMO
F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central γ rotor subunit and the opening and closing of the catalytic ß subunits. Cooperativity in F1-ATPase has long thought to be conferred through the γ subunit, with three key interaction sites between the γ and ß subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the γ axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less γ and the open ß subunit suggests that the complete γ subunit is important for coordinating efficient ATP binding of F1-ATPase.
RESUMO
The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we exploit the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. The conductive state has a canonical cylindrical shaped selectivity filter. The non-conductive state is characterized by flipping of the selectivity filter valine backbone carbonyls to point away from the central axis. The side chain of S620 on the pore helix plays a central role in this process, by coordinating distinct sets of interactions in the conductive, non-conductive, and transition states. Our model represents a distinct mechanism by which ion channels fine tune their activity and could explain the uniquely rapid inactivation kinetics of HERG.
Assuntos
Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Potássio , Humanos , Potássio/metabolismo , Canal de Potássio ERG1/metabolismo , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/química , Canais de Potássio Éter-A-Go-Go/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Cinética , Células HEK293 , Ativação do Canal Iônico , Modelos MolecularesRESUMO
CCCTC-binding factor (CTCF) is an insulator protein that binds to a highly conserved DNA motif and facilitates regulation of three-dimensional (3D) nuclear architecture and transcription. CTCF binding sites (CTCF-BSs) reside in non-coding DNA and are frequently mutated in cancer. Our previous study identified a small subclass of CTCF-BSs that are resistant to CTCF knock down, termed persistent CTCF binding sites (P-CTCF-BSs). P-CTCF-BSs show high binding conservation and potentially regulate cell-type constitutive 3D chromatin architecture. Here, using ICGC sequencing data we made the striking observation that P-CTCF-BSs display a highly elevated mutation rate in breast and prostate cancer when compared to all CTCF-BSs. To address whether P-CTCF-BS mutations are also enriched in other cell-types, we developed CTCF-INSITE-a tool utilising machine learning to predict persistence based on genetic and epigenetic features of experimentally-determined P-CTCF-BSs. Notably, predicted P-CTCF-BSs also show a significantly elevated mutational burden in all 12 cancer-types tested. Enrichment was even stronger for P-CTCF-BS mutations with predicted functional impact to CTCF binding and chromatin looping. Using in vitro binding assays we validated that P-CTCF-BS cancer mutations, predicted to be disruptive, indeed reduced CTCF binding. Together this study reveals a new subclass of cancer specific CTCF-BS DNA mutations and provides insights into their importance in genome organization in a pan-cancer setting.
Assuntos
Fator de Ligação a CCCTC , Aprendizado de Máquina , Mutação , Fator de Ligação a CCCTC/metabolismo , Fator de Ligação a CCCTC/genética , Humanos , Sítios de Ligação/genética , Cromatina/metabolismo , Cromatina/genética , Neoplasias/genética , Neoplasias/metabolismo , Ligação Proteica , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Masculino , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismoRESUMO
F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalytic mechanism and isolated F1-ATPase subcomplexes can also hydrolyze ATP to generate rotation of their central γ rotor subunit. As ATP is hydrolyzed, the F1-ATPase cycles through a series of conformational states that mediates unidirectional rotation of the rotor. However, even in the absence of a rotor, the α and ß subunits are still able to pass through a series of conformations, akin to those that generate rotation. Here, we use cryoelectron microscopy to establish the structures of these rotorless states. These structures indicate that cooperativity in this system is likely mediated by contacts between the ß subunit lever domains, irrespective of the presence of the γ rotor subunit. These findings provide insight into how long-range information may be transferred in large biological systems.
Assuntos
Adenosina Trifosfatases , Trifosfato de Adenosina , Hidrólise , Microscopia Crioeletrônica , Subunidades Proteicas/química , Conformação Proteica , RotaçãoRESUMO
Organic Cation Transporter 1 (OCT1) plays a crucial role in hepatic metabolism by mediating the uptake of a range of metabolites and drugs. Genetic variations can alter the efficacy and safety of compounds transported by OCT1, such as those used for cardiovascular, oncological, and psychological indications. Despite its importance in drug pharmacokinetics, the substrate selectivity and underlying structural mechanisms of OCT1 remain poorly understood. Here, we present cryo-EM structures of full-length human OCT1 in the inward-open conformation, both ligand-free and drug-bound, indicating the basis for its broad substrate recognition. Comparison of our structures with those of outward-open OCTs provides molecular insight into the alternating access mechanism of OCTs. We observe that hydrophobic gates stabilize the inward-facing conformation, whereas charge neutralization in the binding pocket facilitates the release of cationic substrates. These findings provide a framework for understanding the structural basis of the promiscuity of drug binding and substrate translocation in OCT1.
Assuntos
Proteínas de Transporte de Cátions Orgânicos , Transportador 1 de Cátions Orgânicos , Humanos , Transportador 1 de Cátions Orgânicos/genética , Transportador 1 de Cátions Orgânicos/química , Transportador 1 de Cátions Orgânicos/metabolismo , Proteínas de Transporte de Cátions Orgânicos/química , Transporte Biológico , Transportador 2 de Cátion Orgânico/metabolismoRESUMO
The GIGYF proteins interact with 4EHP and RNA-associated proteins to elicit transcript-specific translational repression. However, the mechanism by which the GIGYF1/2-4EHP complex is recruited to its target transcripts remain unclear. Here, we report the crystal structures of the GYF domains from GIGYF1 and GIGYF2 in complex with proline-rich sequences from the miRISC-binding proteins TNRC6C and TNRC6A, respectively. The TNRC6 proline-rich motifs bind to a conserved array of aromatic residues on the surface of the GIGYF1/2 GYF domains, thereby bridging 4EHP to Argonaute-miRNA complexes. Our structures also reveal a phenylalanine residue conserved from yeast to human GYF domains that contributes to GIGYF2 thermostability. The molecular details we outline here are likely to be conserved between GIGYF1/2 and other RNA-binding proteins to elicit 4EHP-mediated repression in different biological contexts.
Assuntos
Proteínas de Transporte , MicroRNAs , Humanos , Proteínas de Transporte/metabolismo , Proteínas de Ligação a RNA/metabolismo , MicroRNAs/metabolismoRESUMO
Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells of convalescent patients using SARS-CoV-2 receptor binding domains carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (S309) by orders of magnitude. They also provide prophylactic and therapeutic in vivo protection of female hACE2 mice against viral challenge. Our results indicate that exposure to SARS-CoV-2 induces antibodies that maintain broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Feminino , Animais , Camundongos , Anticorpos Amplamente Neutralizantes , Leucócitos Mononucleares , Anticorpos Antivirais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Epitopos , Glicoproteína da Espícula de Coronavírus/genética , Testes de NeutralizaçãoRESUMO
F1Fo ATP synthase functions as a biological generator and makes a major contribution to cellular energy production. Proton flow generates rotation in the Fo motor that is transferred to the F1 motor to catalyze ATP production, with flexible F1/Fo coupling required for efficient catalysis. F1Fo ATP synthase can also operate in reverse, hydrolyzing ATP and pumping protons, and in bacteria this function can be regulated by an inhibitory ε subunit. Here we present cryo-EM data showing E. coli F1Fo ATP synthase in different rotational and inhibited sub-states, observed following incubation with 10 mM MgATP. Our structures demonstrate how structural transitions within the inhibitory ε subunit induce torsional movement in the central stalk, thereby enabling its rotation within the Fο motor. This highlights the importance of the central rotor for flexible coupling of the F1 and Fo motors and provides further insight into the regulatory mechanism mediated by subunit ε.
Assuntos
Trifosfato de Adenosina , Escherichia coliRESUMO
Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.
RESUMO
Cells and tissues are routinely cultured in vitro for biological research with findings being extrapolated to their host organ and tissue function. However, most samples are cultured and studied in unphysiological environments, without temporal variation in the biochemical cues that are ubiquitous in vivo. The artificiality of these conditions undermines the predictive value of cell culture studies. We ascribe the prevalence of this suboptimal culture methodology to the lack of practical continuous flow systems that are economical and robust. Here, we design and implement an expandable multiplexed flow system for cell culture superfusion. By expanding on the concept of the planar peristaltic pump, we fabricated a highly compact and multiplexed pump head with up to 48 active pump lines. The pump is incorporated into a custom, open-top superfusion system configured for conventional multi-well culture plates. We then demonstrated the utility of the system for in vitro circadian entrainment using a daily cortisol pulse, generating a sustained circadian amplitude that is essential for physiological emulation and chrono-pharmacological studies. The multiplexed pump is complemented by a package of fluidic interconnection and management methods enabling user-friendly and scalable operation. Collectively, the suite of technologies provides a much-needed improvement in physiological emulation to support the predictive value of in vitro biomedical and biological research.
Assuntos
Técnicas de Cultura de Células , Ritmo Circadiano , Ritmo Circadiano/fisiologiaRESUMO
Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second-generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor-binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that the neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies.
Assuntos
Betacoronavirus/fisiologia , Vacinas contra COVID-19/imunologia , Infecções por Coronavirus/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/metabolismo , Sequência Conservada/genética , Evolução Molecular , Humanos , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ligação Proteica , Domínios Proteicos/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Desenvolvimento de VacinasRESUMO
Epidemic thunderstorm asthma (ETSA) occurs following a thunderstorm due to the interaction of environmental and immunologic factors. Whilst first reported in the 1980s, the world's largest event in Melbourne, Australia, on November 21, 2016 has led to a wealth of clinical literature seeking to identify its mechanisms, susceptibility risk factors, and management approaches. Thunderstorm asthma (TA) typically presents during an aeroallergen season in individuals sensitized to perennial rye grass pollen (RGP) in Australia, or fungus in the United Kingdom, in combination with meteorological factors such as thunderstorms and lightning activity. It is now well recognized that large pollen grains, which usually lodge in the upper airway causing seasonal allergic rhinitis (SAR), are ruptured during these events, leading to sub-pollen particles respirable to the lower respiratory tract causing acute asthma. The identified risk factors of aeroallergen sensitization, specifically to RGP in Australians with a history of SAR, and individuals born in Australia of South-East Asian descent as a risk factor for TA has been key in selecting appropriate patients for preventative management. Moreover, severity-determining risk factors for ETSA-related asthma admission or mortality, including pre-existing asthma or prior hospitalization, poor inhaled corticosteroid adherence, and outdoor location at the time of the storm are important in identifying those who may require more aggressive treatment approaches. Basic treatments include optimizing asthma control and adherence to inhaled corticosteroid therapy, treatment of SAR, and education regarding TA to increase recognition of at-risk days. Precision treatment approaches may be more beneficial in select individuals, including the use of allergen immunotherapy and even biologic treatment to mitigate asthma severity. Finally, we discuss the importance of environmental health literacy in the context of concerns surrounding the increased frequency of ETSA due to climate change and its implications for the frequency and severity of future events.
RESUMO
F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. Isolated F1-ATPase catalytic cores can hydrolyze ATP, passing through six intermediate conformational states to generate rotation of their central γ-subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in the F1-ATPase, the structure of an important conformational state (the binding-dwell) has remained elusive. Here, we exploit temperature and time-resolved cryo-electron microscopy to determine the structure of the binding- and catalytic-dwell states of Bacillus PS3 F1-ATPase. Each state shows three catalytic ß-subunits in different conformations, establishing the complete set of six states taken up during the catalytic cycle and providing molecular details for both the ATP binding and hydrolysis strokes. We also identify a potential phosphate-release tunnel that indicates how ADP and phosphate binding are coordinated during synthesis. Overall these findings provide a structural basis for the entire F1-ATPase catalytic cycle.
Assuntos
ATPases Bacterianas Próton-Translocadoras/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus/enzimologia , ATPases Bacterianas Próton-Translocadoras/genética , ATPases Bacterianas Próton-Translocadoras/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Hidrólise , Mutação , Fosfatos/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Rotação , TemperaturaRESUMO
Cellular mechanical properties (e.g. compressibility) are important biophysical markers in relation to cellular processes and functionality. Among the methods for cell mechanical measurement, acoustofluidic methods appear to be advantageous due to tunability, biocompatibility and acousto-mechanical nature. However, the previous acoustofluidic methods were limited in throughput and number of measurements. In this study, we developed a high-throughput microfluidic compressibility cytometry approach using multi-tilted-angle surface acoustic wave, which can provide thousands of single-cell compressibility measurements within minutes. The compressibility cytometer was constructed to drag microparticles or cells towards the microfluidic channel sidewall at different segments based on their biophysical properties (such as size and compressibility), as a result of the varied balance between acoustics and flow. Mathematical analysis and computational simulation revealed that the compressibility of a cell could be estimated from the position of collision with the sidewall. Microbeads of different materials and sizes were experimentally tested to validate the simulation and to demonstrate the capability to characterise size and compressibility. MDA MB231 cells, of the triple negative breast cancer subtype, were treated with the microtubule disrupting agent colchicine which increased compressibility and treated with the actin disrupting agent cytochalasin B which increased cell size but did not change compressibility. Moreover, the highly metastatic variant MDA MB231 LNm5 cell line showed increased compressibility compared to the parent MDA MB231 cells, indicating the potential utility of high-throughput mechanophenotyping for tumour cell characterisation.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Acústica , Linhagem Celular , Citometria de Fluxo , Microesferas , SomRESUMO
The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1ß monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.
Assuntos
Aterosclerose , Tratamento Farmacológico da COVID-19 , COVID-19 , Fármacos Cardiovasculares , Doenças Cardiovasculares , Endotélio Vascular , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/fisiopatologia , COVID-19/metabolismo , COVID-19/fisiopatologia , Fármacos Cardiovasculares/classificação , Fármacos Cardiovasculares/farmacologia , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/fisiopatologia , Descoberta de Drogas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , SARS-CoV-2RESUMO
Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Especificidade de Anticorpos , Reações Cruzadas , Humanos , Mutagênese Sítio-DirigidaRESUMO
Triple-negative breast cancer (TNBC) has a poor outcome compared to other breast cancer subtypes, and new therapies that target the molecular alterations driving tumor progression are needed. Annexin A1 is an abundant multi-functional Ca2+ binding and membrane-associated protein. Reported roles of Annexin A1 in breast cancer progression and metastasis are contradictory. Here, we sought to clarify the functions of Annexin A1 in the development and progression of TNBC. The association of Annexin A1 expression with patient prognosis in subtypes of TNBC was examined. Annexin A1 was stably knocked down in a panel of human and murine TNBC cell lines with high endogenous Annexin A1 expression that were then evaluated for orthotopic growth and spontaneous metastasis in vivo and for alterations in cell morphology in vitro. The impact of Annexin A1 knockdown on the expression of genes involved in mammary epithelial cell differentia tion and epithelial to mesenchymal transition was also determined. Annexin A1 mRNA levels correlated with poor patient prognosis in basal-like breast tumors and also in the basal-like 2 subset of TNBCs. Unexpectedly, loss of Annexin A1 expression had no effect on either primary tumor growth or spontaneous metastasis of MDA-MB-231_HM xenografts, but abrogated the growth rate of SUM149 orthotopic tumors. In an MMTV-PyMT driven allograft model of breast cancer, Annexin A1 depletion markedly delayed tumor formation in both immuno-competent and immuno-deficient mice and induced epithelial to mesenchymal transition and upregulation of basal markers. Finally, loss of Annexin A1 resulted in the loss of a discrete CD24+/Sca1- population containing putative tumor initiating cells. Collectively, our data demonstrate a novel cell-autonomous role for Annexin A1 in the promotion of tumor-forming capacity in a model of human breast cancer and suggest that some basal-like TNBCs may require high endogenous tumor cell Annexin A1 expression for continued growth.
RESUMO
Chaperonins are biomolecular complexes that assist in protein folding. Thermophilic factor 55 (TF55) is a group II chaperonin found in the archaeal genus Sulfolobus that has α, ß and γ subunits. Using cryo-electron microscopy, structures of the ß-only complex of S. solfataricus TF55 (TF55ß) were determined to 3.6-4.2â Å resolution. The structures of the TF55ß complexes formed in the presence of ADP or ATP highlighted an open state in which nucleotide exchange can occur before progressing in the refolding cycle.